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Does Hypergravity Regulate Gene Expression Of Я1- Integrin?  J. Krishnakumar1,2,3, J. Wilcox1,3, M.Parra1,3, E. Holton3, N. Searby3, R.Globus1, E. Almeida1 , W. Vercoutere1,3   1NASA Ames Research Center, 2California State University,Hayward, 3National Space Grant Foundation

   Exposure to microgravity or decreased mechanical loading of physiological systems in space or on Earth leads to atrophy in mechanosensitive tissue such as the muscle and bone. Increased mechanical loading strengthens these tissues. In vivo and in vitro studies suggest this sensitivity may occur at the cellular level. Our studies indicate that increased gravity-loading may stimulate primary osteoblast bone cell proliferation via matrix-integrin signaling pathways. We have shown focal adhesion complexes increase in size in hypergravity and that the proliferative response of osteoblasts is dependent on collagen type I and fibronectin. This implicates an enhanced Я1-integrin signaling. We hypothesize that increased Я1-integrin signaling could be due to an increase in Я1-integrin surface expression. This could be due to either 1) sequestered proteins pre-existing in cytosol which move to the cell surface or 2) an increase in Я1-integrin gene expression. To test this, we used cell culture centrifugation (Incu-fuge) to provide hypergravity mechanostimulation and examined Я1-integrin gene expression. The 10-50-g range we use also mimics physiological intermedullary pressure (1.2 — 5 kPa).  For our study we exposed bone cells to constant 25-g for 24hrs. The primary osteoblasts were exposed to 25-g in both collagen coated and uncoated T-25 flasks. To quantify Я1-integrin gene expression Reverse Transcriptase - Polymerase chain Reaction (RT-PCR) was performed on a real time PCR machine. For internal standards cyclophilin was included as the housekeeping gene. From relative mRNA levels Я1-integrin expression for bone cells grown on collagen at 1-g was 26x higher than Я1-integrin expression for bone cells grown on a surface without matrix. Я1-integrin expression in hypergravity increased 3 fold for collagen coated cells. Я1-integrin expression in hypergravity also increased significantly for uncoated cells, but remained well below the basal expression level on 1-g collagen.